Platelets, and the components which they secrete upon activation, play a major role in hemostasis, thrombosis and the development of atherosclerosis (see Petersdorf, R. G., et al., eds., Principles of Internal Medicine, 10th edition, 1983, McGraw-Hill, New York, pp. 292-294 and 1468). Megakaryocytes in the bone marrow form platelets by pinching off pieces of their cytoplasm.
Upon injury to a tissue, blood platelets adhere to the exposed subendothelial tissue through adhesive platelet components. These components also promote platelet-platelet interactions and smooth muscle cell proliferation in response to platelet-derived growth factors. Platelets adhere to other membranes through their membrane protein GPIb to von Willebrand factor (a component of clotting factor VIII) in the subendothelial matrix. This is followed by platelet clot formation by interactions between GPIIb/IIIa, as well as von Willebrand factor, platelet factor 4 and fibrinogen which are secreted from the platelet alpha granules into the interstitial spaces of the clot. Thus, the efficacy of platelet participation in normal processes, as well as in atherosclerosis, is largely dependent upon adequate numbers of platelets and adequate concentrations of the participating components in the platelet membranes and granules.
Platelet production is controlled by sequential regulation of the component steps of megakaryocytopoiesis: 1) commitment of pluripotent stem cells to the megakaryocyte lineage, 2) proliferation of the committed stem cells, 3) polyploidization, 4) cytoplasmic maturation, and 5) platelet release. Greenberg-Sepersky, S. M., et al., Thrombo. Res. 24:299-306 (1981). However, the process of platelet production which occurs at the level of differentiation and maturation of the parent megakaryocytes in the bone marrow is poorly understood.
To date, several humoral factors have been postulated to regulate the steps of megakaryocytopoiesis in vivo and in vitro. In colony-forming assays, which measure the proliferation of committed stem cells, megakaryocyte colony-stimulating factor (Meg-CSF), megakaryocyte potentiator (MK-POT), interleukin-3 (IL-3), interleukin-1 (IL-1), erythropoietin (EPO), and granulocyte-macrophage colony-stimulating factor (GM-CSF), all increase the number and size of megakaryocyte colonies in vitro. Thrombocytopenic serum, a source of the uncharacterized factor "thrombopoietin," or the conditioned medium from bone marrow cultures and cultured human embryonic kidney cells increases the number, ploidy, and size of megakaryocytes in vitro, and the incorporation of radiolabelled precursors into the membrane and alpha granules of newly-released platelets in vivo.
A major limitation of most of these studies is the lack of a purified megakaryocytic cell system. All of the studies which examine megakaryocyte differentiation utilize bone marrow cell preparations. However, except for studies which utilize primary cultures of a single bone marrow cell type, interpretation of the results is complicated by the presence of non-megakaryocytic cells which may act as accessory cells.
The mechanisms controlling thrombopoiesis are not well understood due to the inability to isolate megakaryoblasts away from other bone marrow cells in the absence of accessory cells, and due to the unavailability of a cultured cell line which can serve as a model of the differentiating megakaryocyte. Megakaryocyte differentiation and maturation is characterized by increased polyploidization and enhanced expression of platelet membrane proteins such as GPIb, GPIIb/IIIa and platelet-specific alpha granule formation.
Cell lines which display the characteristics of megakaryocytic cell have been reported. However, these cell lines are limited in their ability to be used as models of megakaryocyte differentiation. For example, MEG-01 cells have been reported to be a megakaryoblastic cell line. However, MEG-01 cells contain the important platelet marker antigen, GPIb, only in the cytoplasm of a subpopulation of larger MEG-01 cells rather than uniformly expressing it on the surface of all the cells. Ogura, M., et al., Blood 66:1384-1392 (1985).
LAMA-84 cells are a megakaryocytic cell line which expresses the platelet marker proteins GPIIb/IIIa. However, LAMA-84 cells do not express the platelet marker protein GPIb. In addition, the LAMA cell line is not committed to the megakaryocytic lineage, but rather represents an earlier stage in differentiation as shown by the fact that they are a tripotent, megakaryocytic, erythroid, and granulocytic cell line. Seigneurin, D., et al., Exp. Hematol. 15:822-832 (1987).
The mutant human megakaryocytic cell line, the HEL cell, does not express the beta subunit for GPIb platelet marker protein and contains an abnormally glycosylated alpha subunit for GPIb. Kieffer, et al., J. Biol. Chem. 261:15854-15862 (1986); Martin et al., Science 233-1235 (1982); Tabilio, A., et al. EMBO J. 3:453-459 (1984).
A promyelocytic leukemic cell line, HL60, responds to inducers of platelet synthesis such as 12-0-tetradecanoyl phorbol 2-acetate (TPA). However, HL60 cells respond to TPA by differentiating to either monocytes or granulocytes instead of inducing platelet production. Michalevicz, R., et al., Leuk. Res. 9:441-448 (1985).
Morgan, D. A., et al., J. Cell. Biol. 100:565-573 (1985) reported a series of human cell lines with properties of megakaryocytes which were isolated and cultured from peripheral blood. However, none of these cell lines are capable of differentiating to a cell with the characteristics of platelet late differentiation morphology, such as alpha granule formation.
The megakaryocytic cell line EST-IU expresses the platelet marker proteins GP IIb/IIIa on its membrane. Sledge, G. W.,et al., Cancer Res. 46:2155-2159 (1986). However, this cell line routinely dies after six months of continuous cell culture (30-35 cell divisions).
Thus, there remains a need for a purified megakaryocytic cell population, in which the culture conditions can be carefully manipulated and the results easily monitored, to study the process of megakaryocytopoiesis, to evaluate the effects of megakaryocytopoietic, hemopoietic and nonhemopoietic factors on the megakaryocyte system, to study platelet formation and release from the parent megakaryocyte (thrombopoiesis), to provide a source for the purification of megakaryocyte and platelet components, to identify new megakaryopoiesis factors from crude preparations and to serve as an assay system for the subsequent isolation and characterization of those new factors.